The clearance of low density lipoprotein (LDL) and very low density lipoprotein (VLDL) by LDL receptor (LDLR) mediated endocytosis plays a critical role in preventing atherosclerosis and vascular disease. The recent finding that the adaptor protein, ARH, binds to the FDNPVY sequence on the LDLR and is required for LDL clearance has provided a tool with which to characterize the molecular mechanisms by which the LDLR mediates lipoprotein uptake and clearance. Recent studies and our preliminary data indicate that ARH plays a complex role in LDLR function. Our hypothesis is that ARH is principally involved in the uptake of LDL and that ARH functions at three levels in the uptake process: first, ARH promotes the ability of the LDLR to bind LDL;second, ARH promotes the uptake of LDLR-LDL complexes;and third, ARH ensures efficient release of LDL from the LDLR in endosomes. This grant proposal will employ a multidisciplinary approach using biochemistry, microscopy and animal studies to identify the mechanisms by which ARH promotes the binding, uptake and degradation of lipoproteins. Experiments will identify the regions of ARH that are responsible for promoting LDL binding, characterize the role of ARH in LDL and VLDL uptake, and determine the function of ARH in the endosomal trafficking of LDL and the LDLR. We will also examine the role of novel ARH binding partners in LDLR function. ARH is a multifunctional adaptor protein and different subsets of binding partners may participate at different stages in the lipoprotein binding and uptake pathway. The characterization of the function of ARH and its associated proteins in lipoprotein uptake by LDLR may clarify our understanding of the clearance process.